Precision peak matching in liquid chromatography-mass spectroscopy

ABSTRACT

A method that identifies common peaks among unidentified peaks in the data from different LC-MS or LC-MS/MS runs is provided. The method employs an algorithm, herein referred to as “Precision Peak Matching (PPM).” The different runs can be from different laboratories, instruments, and biological samples that result in a significant variability in the data. PPM allows estimation and control of precision, defined as the fraction of truly identical peptide pairs among all pairs retrieved, in the matching process. PPM finds the maximal number of peptide pairs at a prescribed precision, thereby allowing quantitative control over the trade off between the number of true pairs missed, and false pairs found. PPM finds common peptides from a database of LC-MS runs of heterogeneous origins, and at the specified precision. PPM fills a much-needed role in proteomics by extracting useful information from disparate LC-MS databases in a statistically rigorous and interpretable manner.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.12/547,874, filed Aug. 26, 2009 the entire content and disclosure ofwhich is incorporated herein by reference.

BACKGROUND

The present invention generally relates to methods of matching peaks indatasets from a plurality of liquid chromatography-mass spectroscopy andapparatuses for the same.

Liquid chromatography-mass spectroscopy (LC-MS) is an analyticalchemistry technique that combines the physical separation capabilitiesof liquid chromatography with the mass analysis capabilities of massspectrometry. Optionally, LC-MS may employ tandem mass spectroscopy(MS/MS), in which multiple mass spectrometry steps are employed with atleast one intervening fragmentation step between the multiple massspectroscopy steps. Liquid chromatography in combination with tandemmass spectroscopy is typically referred to as liquidchromatography-tandem mass spectroscopy (LC-MS/MS), and is a subset ofLC-MS.

Data from a liquid chromatography-mass spectroscopy is typicallygenerated as “features” in a multi-dimensional space including amass-to-charge ratio of a detected material as one axis and a retentiontime of the detected material as another axis. The retention time is thetime it takes for a material to travel through a capillary column thatleads into a vacuum environment in which the material is ionized fordetection by a mass spectrometer. The mass-to-charge ratio is the ratioof the mass of the material to the electrical charge of the material asdetected by a mass spectrometer after the material is ionized in avacuum environment. In its simplest form, a feature is simply a peak inthe LC-MS chromatogram, but a feature may also be a monoisotopic massdeduced from an isotope series, with corresponding retention time andoptional intensity.

Multiple LC-MS runs result in multiple datasets, in which each datasetincludes a list of peaks from one LC-MS run. The list of peaks isrepresented in the multi-dimensional space of a mass-to-charge ratio, aretention time, and optionally, an intensity of the peak. It is achallenge to compare proteomics data from different LC-MS experimentsbecause not all the peaks coincide with corresponding peaks from otherruns in the multi-dimensional space of the mass-to-charge ratio and theretention time.

There is an increasing need for computational methods to compare proteinexpression measured by LC-MS or LC-MS/MS proteomics experiments. Publicdomain proteomic databases such as the Open Proteomic Database andPeptideAtlas have accumulated thousands of LC runs from variouslaboratories, and the numbers continue to increase. Comparisons ofmultiple proteomic experiments based on identified proteins and peptidesare feasible, but limited because most LC-MS or LC-MS/MS peaks areunidentified and therefore overlooked. In addition, many peaks inLC-MS/MS are unidentified because peptide identification by MS/MS ionsearch is still a low percentage sampling process with imperfectreproducibility.

Without sequence information, the common practice is to match peptidesbetween different runs based solely on similarity in mass and normalizedretention time. However, this method is prone to some level ofmismatches because different peptides may share similar mass andnormalized retention times by chance. Thus, peptide matching based onmass and retention time similarity should be accompanied by error rateestimation, especially for complex protein mixture.

The error rate in matching is largely overlooked in the literature. Someof the few references that consider the error rate in matching includeJaffe, J. D. et al., “PEPPeR, a platform for experimental proteomicpattern recognition,” Mol. Cell. Proteomics 5, 1927-1941 (2006), Monroe,M. E. et al., “VIPER: an advanced software package to supporthigh-throughput LC-MS peptide identification,” Bioinformatics 23,2021-2023 (2007), and Anderson, K. K., Monroe, M. E. & Daly, D. S.,“Estimating probabilities of peptide database identifications toLC-FTICR-MS observations,” Proteome Sci 4, 1 (2006). The PEPPeR pipelineestimates the mismatching rate by bootstrapping, while VIPER estimatesthe probability of correct matching by Expectation Maximization (EM).VIPER uses Accurate Mass and Time Tag (AMT) peptide identification,which matches mass and retention time pairs to a database of identifiedpeptides. VIPER estimates the mismatching rate by searching against thedatabase of identified peptides where every mass is shifted by aconstant amount, such as 7 Dalton (Da.). However, for both PEPPeR andVIPER, the accuracy of the estimated mismatching rates is unclear, andrequire some peptides to be identified. More importantly, both arelimited to comparison among similar proteomic experiments.

BRIEF SUMMARY

In an embodiment of the present invention, a method that identifiescommon peaks among unidentified peaks in the datasets from differentLC-MS or LC-MS/MS runs is provided. The method employs an algorithm thatis herein referred to as “Precision Peak Matching,” or “PPM.” Thedifferent runs can be from different laboratories, instruments, andbiological samples that result in a significant variability in the data.PPM can be employed during matching of two peak lists from two differentLC-MS runs.

According to an aspect of the present invention, a system for matchingpeaks in liquid chromatography-mass spectroscopy (LC-MS) datasets frommultiple runs is provided. The system includes a memory and a processordevice in communication with the memory. The system is configured toperform a method including generating, by employing the processor andthe memory, an aligned query list for peaks from a first dataset from afirst LC-MS run; generating, by employing the processor and the memory,a target peak list for peaks from a second dataset from a second LC-MSrun; generating, by employing the processor and the memory, amass-to-charge ratio (m/z) tolerance parameter and a retention time (Rt)tolerance parameter that satisfy a specification input criterion for afalse matching rate between the aligned query list and the target peaklist; determining, by employing the processor and the memory, a truematching rate between the aligned query list and the target peak listemploying the m/z tolerance parameter and the Rt tolerance parameter;selecting, by employing the processor and the memory, an optimized m/ztolerance value and an optimized Rt tolerance value by repeating thestep of selecting the m/z tolerance parameter and Rt tolerance parameterand the step of determining the true matching rate; and generating, byemploying the processor and the memory, an optimal list of matches amongpeaks across the aligned query list and the target peak list employingthe optimized m/z tolerance value and the optimized Rt tolerance valueas matching parameters.

According to another aspect of the present invention, a method formatching peaks in liquid chromatography-mass spectroscopy (LC-MS)datasets from multiple runs is provided. The method includes generating,by employing the processor and the memory, an aligned query list forpeaks from a first dataset from a first LC-MS run, generating, byemploying the processor and the memory, a target peak list for peaksfrom a second dataset from a second LC-MS run; generating, by employingthe processor and the memory, a mass-to-charge ratio (m/z) toleranceparameter and a retention time (Rt) tolerance parameter that satisfy aspecification input criterion for a false matching rate between thealigned query list and the target peak list; determining, by employingthe processor and the memory, a true matching rate between the alignedquery list and the target peak list employing the m/z toleranceparameter and the Rt tolerance parameter; selecting, by employing theprocessor and the memory, an optimized m/z tolerance value and anoptimized Rt tolerance value by repeating the step of selecting the m/ztolerance parameter and Rt tolerance parameter and the step ofdetermining the true matching rate; and generating, by employing theprocessor and the memory, an optimal list of matches among peaks acrossthe aligned query list and the target peak list employing the optimizedm/z tolerance value and the optimized Rt tolerance value as matchingparameters.

According to yet another aspect of the present invention, amachine-readable data storage device embodying a program ofmachine-executable instructions to match peaks in liquidchromatography-mass spectroscopy (LC-MS) datasets from multiple runs isprovided. The program includes the method that the system is configuredto perform as described above.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic representation of a peak list or a feature list intwo dimensions containing annotated and non-annotated features.

FIG. 2 is a schematic representation of peaks with true match and peakswith false match in the two-dimensional space in which the x-axis is apairwise mass difference and the y-axis is a pairwise retention timedifference.

FIG. 3 is a graph schematically illustrating mass shifting employed toeffect a false match.

FIG. 4 is a graph illustrating that estimated precision according to thepresent invention matches true precision more closely than estimatedprecision provided by shuffle methods.

FIG. 5 is a first flow chart illustrating a method for determining afalse matching rate once an m/z matching tolerance and an Rt matchingtolerance are set.

FIG. 6 is a second flow chart illustrating a method for determining atrue matching rate when an m/z matching tolerance and an Rt matchingtolerance are provided.

FIG. 7 is a third flow chart illustrating a method for determining anm/z matching tolerance and an Rt matching tolerance at a specifiedtarget false matching rate and a specified false matching ratetolerance.

FIG. 8 is a fourth flow chart illustrating a method for generating anoptimal list of matches once the optimal m/z tolerance value and theoptimal Rt tolerance value are determined.

FIG. 9 illustrates an exemplary system according to an embodiment of thepresent invention.

DETAILED DESCRIPTION

As stated above, the present invention relates to methods of matchingpeaks in datasets from a plurality of liquid chromatography-massspectroscopy and apparatuses for the same, which are now described indetail with accompanying figures. Throughout the drawings, the samereference numerals or letters are used to designate like or equivalentelements. The drawings are not necessarily drawn to scale.

As used herein, “liquid chromatography-mass spectroscopy” (LC-MS) is anytechnique that employs liquid chromatography and any type of massspectroscopy.

As used herein, a “liquid chromatography-mass spectroscopy apparatus,”or an “LC-MS apparatus” is any apparatus that generates data on LC-MS ofa sample.

As used herein, an “LC-MS run” is an experiment on an LC-MS apparatusthat generates an “LC-MS dataset,” i.e., a dataset representing theLC-MS data from the experiment.

As used herein, a “liquid chromatography-mass spectroscopy database,” oran “LC-MS database” is any database that stores at least one datasetfrom any LC-MS runs.

As used herein, a “mass-to-charge ratio,” or an “m/z” is the ratio of amass to a charge of an ionized molecule or an ion as detected by a massspectrometer in an LC-MS apparatus.

As used herein, a “retention time” is the time that a capillary columnretains a particular compound that is detected as a peak in an LC-MSrun. The retention time is the time between the starting time at whichdiffusion of a sample along a capillary column and the time at which anionized molecule or an ion is detected by a mass spectrometer in anLC-MS apparatus.

As used herein, a “peak” is a data point from an LC-MS run andrepresents presence of at least one compound in the material of thesample of the LC-MS run at a mass-to-charge ratio and at a retentiontime. Each peak in an LC-MS dataset has a unique combination of valuesfor its mass-to-charge ratio and its retention time.

As used herein, a “peak list” or a “feature list” is a list of peaksfrom at least one LC-MS run.

As used herein, an “annotated peak” is a peak for which the identity ofthe material represented by the peak is known.

As used herein, an “annotated peak list” is a list of annotated peaks.

As used herein, a “non-annotated peak” is a peak for which the identityof the material represented by the peak is unknown.

As used herein, a “non-annotated peak list” is a list of non-annotatedpeaks.

As used herein, a “mass shift” refers to shifting of every mass in thedata from one LC-MS run by a number having a unit of Da. The number canbe an integer.

As used herein, a “course grain search” refers to a search method inwhich a dataset is divided into large subsets. A course grain search ona dataset employs a fewer number of subsets than a fine grain search onthe same dataset.

As used herein, a “fine grain search” refers to a search method in whicha dataset is divided into small subsets. A fine grain search on adataset employs a greater number of subsets than a course grain searchon the same dataset.

As used herein, a “memory” refers to a device, an apparatus, or amanufactured physical structure that is configured to store informationand allow retrieval of the information.

As used herein, a “processor device” refers to a device, an apparatus,or a manufactured physical structure that includes an electronic circuitfor processing data.

Referring to FIG. 1, a schematic representation of a peak list 10, or afeature list, in two dimensions illustrates the data structure includedin a peak list from an LC-MS run. One axis is a mass-to-charge ratio(m/z) axis. The other axis is a retention time (Rt) axis. Annotatedpeaks 20, i.e., annotated features, are shown as squares. The identityof the compound representing each annotated peak is known. Non-annotatedpeaks 30, i.e., non-annotated features, are shown in circles. Theidentity of the compound representing each non-annotated peak isunknown. Each peak can be represented as a two dimensional data point.Each peak can be represented a two-dimensional vector including twocomponents, or can be represented by a point in a two-dimensionalCartesian coordinate.

In an embodiment of the present invention, a method that identifiescommon peaks among unidentified peaks in the datasets from differentLC-MS or LC-MS/MS runs employs an algorithm, which is herein referred toas “Precision Peak Matching” or “PPM.” The different runs can be fromdifferent laboratories, instruments, and biological samples that resultin a significant variability in the data. PPM allows estimation andcontrol of precision, defined as the fraction of truly identical peptidepairs among all pairs retrieved, in the matching process. PPM finds themaximal number of peptide pairs at a prescribed precision, therebyallowing quantitative control over the trade off between the number oftrue pairs missed, and false pairs found. PPM can find common peptidesfrom a database of LC-MS runs of heterogeneous origins at a specifiedprecision. PPM can fill a much-needed role in proteomics by extractinguseful information from disparate LC-MS databases in a statisticallyrigorous and interpretable manner.

PPM can be employed to match any LC-MS data including data on peptides.PPM can extend the current peptide matching methods based on mass andretention time by estimating and controlling the mismatching rate,enabling it to compare not only similar LC-MS or LC-MS/MS runs, but alsoruns from different laboratories, instruments, and biological samples.PPM seeks the maximal number of matched peak pairs under a prescribedmismatching rate, and the estimation of this mismatching rate isessential to its strategy. PPM also estimates the precision of peakmatching, defined as the fraction of matched peak pairs that are correct(i.e., identical), by matching two runs where the mass of one run isshifted.

PPM can find the maximal number of matched peaks between two runs at aprescribed precision. For each mass tolerance A and retention timetolerance B, PPM estimates the actual precision P by treating the numberof matches with mass shift as false matches, denoted as M. Denote N asthe number of matches without mass shift, then P=1−M/N. By searchingover the parameter space of A and B, PPM maximizes N with the constraintthat P is not below a prescribed precision.

PPM estimates the precision of peak matching, defined as the fraction ofmatched peak pairs that are correct (i.e., identical), by matching tworuns where the mass of one run is shifted by a “mass shift” definedabove. This approach is herein referred to as “mass shifting.” Forexample, the mass of one LC-MS dataset can be shifted between 3 Da and200 Da, although lesser and greater shifts can also be employed.

A formal description of PPM is provided herein. Let P denote theprecision of peptide matching between runs j and l, each with nj and nlpeaks, respectively, at mass tolerance A and retention time tolerance B.Let N denote the total number of matched peak pairs between j and l, Mthe total number of mismatches between them with a mass shift of dm, andm_(ij) and t_(ij) as the mass and retention time of the ith peak in runj, respectively. Then,

${N = {\sum\limits_{i = 1}^{nj}{\sum\limits_{l = 1}^{nl}{f\left( {m_{ij},{t_{{ij},}m_{{kl},}t_{kl}},A,B} \right)}}}},{M = {\overset{nj}{\sum\limits_{i = 1}}{\sum\limits_{l = 1}^{nl}{f\left( {{m_{ij} + {d\; m}},{t_{{ij},}m_{{kl},}t_{kl}},A,B} \right)}}}},$

wherein the matching function ƒ( ) is defined as:

$\begin{matrix}{{{f\left( {{m_{{ij},}t_{{ij},}m_{{kl},}t_{kl}},A,B} \right)} = 1},{{{{if}\mspace{14mu} \left( \frac{m_{ij} - m_{kl}}{A} \right)^{2}} + \left( \frac{t_{ij} - t_{kl}}{B} \right)^{2}} \leq 1},} & {{Equation}\mspace{14mu} (1)}\end{matrix}$

and ƒ( )=0 otherwise.

Then the precision P is defined as:

$\begin{matrix}{{{P\left( {j,l,A,B} \right)} = {1 - \frac{M}{N}}},} & {{Equation}\mspace{14mu} (2)}\end{matrix}$

with its floor set at 0 and ceiling set at 1. Given a prescribedprecision level P₀, PPM becomes the following optimization problem ofmaximizing N with variable A, B:

$\begin{matrix}{{{Argmax}_{{A \in {({0, \propto})}},{B \in {({0, \propto})}}}{\sum\limits_{i = 1}^{nj}{\sum\limits_{l = 1}^{nl}{f\left( {m_{ij},{t_{{ij},}m_{{kl},}t_{kl}},A,B} \right)}}}},} & {{Equation}\mspace{14mu} (3)}\end{matrix}$

subject to the constraint that P(j, l, A, B)≧P₀. “Arg max” stands for“argument of the maximum,” that is to say, the set of points of thegiven argument for which the value of the given expression attains itsmaximum value. Thus, the arg max function generates the maximum possiblevalue under the conditions allowed to the arguments.

PPM is implemented by a systematic search strategy to find the optimalmass and retention time tolerance, which uses coarse grain search firstfollowed by fine grain search. The input for PPM is two aligned peaklists from two LC-MS or LC-MS/MS runs, where each peak has a mass,retention time and intensity value. To correct for retention time driftbetween runs, the peak lists are assumed to be already aligned againsteach other. The output from PPM is a list of peak pairs where each pairof peaks are from two runs and are of the same chemical species, at aprescribed precision level. The number of such peak pairs is maximizedby PPM under the constraint of the prescribed precision level.

Referring to FIG. 2, peaks with true match and peaks with false matchfrom two LC-MS runs are schematically illustrated in the two-dimensionalspace in which the x-axis is the pairwise mass difference and the y-axisis the pairwise retention time difference. True matches do not require amass shift to produce a match. False matches require a mass shift toproduce a match. True matches are shown in large open diamond symbolsand false matches are shown as small filled diamond symbols,respectively. PPM works by finding the optimal boundary of similaritycutoffs in mass and retention time.

The boundary is a closed line in the two-dimensional space. PPM canemploy an ellipse decision boundary found by PPM. In this case, PPM usesan ellipse boundary around the origin. Peak pairs within the boundaryare considered to be the same chemical species. PPM can adjust the longand short semiaxes of the ellipse during optimization. To achieve theprescribed precision, PPM estimates the precision for any ellipseboundary using mass shift. An exemplary ellipse decision boundary thatprovides a precision P of 0.95 and another exemplary ellipse boundarythat provides a precision P of 0.90 are shown. PPM can also employ anyother type of shapes for the boundary, including a rectangle in thetwo-dimensional space in which the x-axis is a pairwise mass differenceand the y-axis is a pairwise retention time difference.

Referring to FIG. 3, a graph schematically illustrates mass shifting. Inan exemplary peak matching employing the method of an embodiment of thepresent invention, peptide matching based on similarity in mass andretention time for two LC-MS runs was performed. In this exemplary peakmatching, two peptides from two runs are considered a match if theirmasses are within 0.2 Da of each other's mass and a retention timedifference is within 60 scans. The peaks from the two runs are plottedtogether. The arrows (total 24) connect matched peptides. The mass ofsome of the peptides in the second run with each mass was incremented by3 Da to effect the match.

The precision within an ellipse boundary can be estimated by shiftingevery mass of one run by a small integer amount. Within the boundary, ifthe total number of matches is defined as T, and the number of falsematches is defined as NF, then the precision of match can be estimatedas 1−(NF/T). In FIG. 3, the precision is 1−(5/24)=79%. Furthermore, theerror bars of the precision can be estimated by using multiple massshifts.

Because PPM can estimate the precision for every ellipse boundary, itcan then adjust the long and short semiaxes of the ellipse to maximizethe total number of matches. The total number of matches can berepresented, for example, by the total number of points within aboundary ellipse as illustrated in FIG. 2, subject to the constraintthat the estimated precision is not less than the prescribed precisionlevel. The constrained optimization problem can be solved by usingcoarse grain search first, followed by a fine grain search around thepreviously found minima. Depending on the data, there is no guaranteethat the desired boundary exist. Error bars of the precision can beuseful guides, especially when the peaks are sparse.

FIG. 4 is a graph illustrating that estimated precision according to anembodiment of the present invention matches true precision more closelythan estimated precision provided by shuffle methods. Precisionestimated by mass shifting is consistent with actual precision. FIG. 4is a graph of Precision vs. A, in which A is the mass tolerance for thefirst two runs of an 18-mix sample, i.e., a sample having 18 compounds.B is the retention time tolerance, and is set to be proportional to A ata ratio of 300 scans/Da. The curve labeled “est P” represents precisionP as defined above, and is calculated by using mass shifts of 10, −10,11, and −11 Da. Error bars represent one standard deviation (SD). Thecurve labeled “shuffle P” is a curve that is calculated by randomlyshuffling the retention times of one run with masses fixed, and countingthe number of matches as mismatches in the manner known in the art.Methods of an embodiment of the present invention can improve estimationof precision of matches more accurately than prior art methods.

Because PPM can compare two LC-MS runs by adjusting the mass andretention time tolerance to fit prescribed precision, it can be a searchtool to compare a query run against a database of runs, which can besimilar runs in the same experiment, historical runs from the same ordifferent laboratories. In the course of the research leading to thepresent invention, it has been demonstrated that PPM can work withheterogeneous runs from different labs.

Thus, mass shifting with appropriate shift amount can help estimateprecision in peak matching. PPM maximizes the number of matches at aprescribed precision by adjusting the values of mass and retention timetolerance on the fly. PPM can serve as a search tool to compare a queryrun against a database of runs for finding common peptides or similarruns, including runs of heterogeneous origin. Methods of employing PPMto find matching peaks from two different LC-MS runs are describedemploying various flow charts.

Referring to FIG. 5, a first flow chart 100 illustrates a method fordetermining a false matching rate once an m/z matching tolerance and anRt matching tolerance are set. Referring to step 120, a first dataset isprovided, for example, from an LC-MS apparatus. The first dataset can befetched from the LC-MS apparatus to a computing device such as acomputer, for example, by electronic transmission or via a data storagemedium such as a flash drive, a CD ROM, or a DVD ROM. The computingdevice can include a memory and a processor device in communication withthe memory. Alternately, the first dataset can be fetched from an LC-MSdatabase. Referring to step 121, a second dataset is provided, forexample, from an LC-MS database. The second dataset can be fetched fromthe LC-MS database to the computing device via electronic transmissionemploying any transmission protocols, e.g., over the internet. The LC-MSdatabase from which the second dataset is fetched can be the LC-MSdatabase from which the first dataset is fetched, or can be a differentLC-MS database. Alternately, the second dataset can be fetched from anLC-MS apparatus. The first and second datasets are fetched from the sameLC-MS apparatus or different LC-MS apparatuses.

Referring to step 101, a query peak list is generated from the firstdataset. The query peak list includes only the peaks for which thematching peaks are to be subsequently searched within the seconddataset. The query list can include all of the peaks in the firstdataset, or can include a subset of the first dataset that is less thanthe first dataset. The query list may be a subset of the original peaksbased on intensity, m/z or Rt.

Referring to step 102, a target peak list is generated from the seconddataset. The target peak list is a list of peaked to be queried for inthe second dataset. There can be a one-to-one correspondence between allof the peaks in the target peak list and all of the peaks in the querypeak list generated at step 101.

Referring to step 111, an aligned query list is generated from the querypeak list. The query peak list is aligned to correct for retention timedrift between LC-MS runs. The alignment process can provide a linearcompensation for retention time to the peaks in the query peak listwithin a range of retention time as provided in the first dataset. Aprogram storage device 180 can provide program instructions and/or aparameter set for effecting the generation of the aligned query list. Inone embodiment, the target peak list generated at step 102 can providedata to be used as an alignment parameter.

Referring to step 113, a shifted target list is generated by performingat least one mass shift to the target peak list. The program storagedevice 180 can provide program instructions and/or a parameter set foreffecting the generation of the shifted target list. The shifted targetlist can include peaks that are mass-shifted, i.e., peaks on which amass shift is performed. In one embodiment of the present invention, allcharges can be assumed to be a unit charge in the mass-to-charge ratiodata. The mass shift can be performed for mass-to-charge ratio if themass cannot be separated from the charge. The amount(s) of mass shiftcan be predetermined, or can be determined based on the nature of thefirst dataset and the second dataset. The amount of mass shift can be,for example, from 3 Da to 200 Da, although lesser and greater massshifts are also contemplated herein. Integer mass shifts may bepreferable since peptide features tend to be spaced at intervals ofapproximately 1 Da.

The peaks in the shifted target list can be mass-shifted by the samemass-differential or by the same mass-to-charge differential betweeneach pair of an original peak and a shifted peak. In this case, theamount of mass shift is recorded and the shifted target list can includea two-dimensional vector for each shifted peak, in which thetwo-dimensional vector includes a shifted mass-to-charge ratio componentand a retention time component.

The shifted target list can have multiple mass-shifted from eachoriginal peak such that each mass-shifted peak from the same originalpeak is shifted by different mass-to-charge differentials or bydifferent mass differentials between each pair of the original peak andone of the shifted peaks. The shifted target list can include athree-dimensional vector for each shifted peak, in which thethree-dimensional vector includes a shifted mass-to-charge ratiocomponent, a retention time component, and a third componentrepresenting the amount of mass shift or the amount of mass-to-chargeshift.

Referring to step 160, peaks are matched between the aligned query listand the shifter target list. The “Precision Pair Matching” (PPM) methoddescribed above is employed to perform peak matching between the alignedquery list generated at step 111 and the shifted target list generatedat step 113. The program storage device 180 can provide programinstructions and/or a parameter set for effecting PPM. In oneembodiment, the program storage device can provide a value for amass-to-charge ratio (m/z) tolerance parameter and a value for aretention time (Rt) tolerance parameter. Alternately, the PPMparameters, i.e., a value for the m/z parameter and a value for Rtparameter, can be provided externally. For example, step 150 and step151 can be employed to manually input a value for the m/z toleranceparameter and a value for the Rt tolerance parameter, respectively.

Referring to step 170, a false match list is generated based on thematching of peaks between the aligned query list and the shifter targetlist. The algorithm of PPM as described above determines false matches,i.e., matches that require a mass shift. The algorithm generates a falsematch list, i.e., a list of false matches as illustrated above by anexample in FIG. 2. Step 113, step 160, and step 170 are collectivelyreferred to as step 190, in which the false matching rate is determinedbased on the aligned query list from step 111 and the target peak listfrom step 102.

Referring to step 190, the false match rate is calculated based on thefalse match list from step 170. The false matching rate is determinedunder the condition of the given value of the m/z parameter and thegiven value for the Rt parameter, which are provided either by theprogram storage device 180 or by step 150 and 160. As discussed above,the false matching rate is a ratio of a total number of matches thatrequires a mass shift to achieve a match to a total number of matches inpeaks. The false matching rate provided as an output of the first flowchart 100.

Referring to FIG. 6, a second flow chart 200 illustrates a method fordetermining a true matching rate when an m/z matching tolerance and anRt matching tolerance. The second flow chart 200 can employ step 111 forgenerating an aligned query list, step 102 for generating a target peaklist, step 150 for providing a manual input for the m/z toleranceparameter, step 151 for providing a manual input for the Rt toleranceparameter, and the program storage device 180 that are described above.

Referring to step 201, the aligned query list from step 111 is splitinto an annotated query list and a non-annotated query list. Thenon-annotated query list is generated, and can be stored, at step 212.The annotated query list is generated at step 211, and is forwarded tostep 260.

Referring to step 203, the target peak list from step 102 is split intoan annotated target list and a non-annotated target list. Thenon-annotated target list is generated, and can be stored, at step 214.The annotated query list is generated at step 213, and is forwarded tostep 260.

Referring to step 260, peaks are matched between the annotated querylist and the annotated target list employing the method of PPM asdescribed above. The peaks are matched only for true matches, i.e., formatches that do not require any mass shift. False matches, i.e., matchesthat require a mass shift, are discarded at this step.

Referring to step 270, a true match list is generated based on thematching of peaks between the annotated query list and the annotatedtarget list.

Referring to step 280, the true matching rate is determined from thetrue match list. The true matching rate is a ratio of a total number ofmatches that does not require a mass shift to achieve a match to a totalnumber of matches in peaks.

Referring to FIG. 7, a third flow chart 300 illustrates a method fordetermining an m/z matching tolerance and an Rt matching tolerance at aspecified target false matching rate and a specified false matching ratetolerance. The method of the third flow chart 300 employs methodsdescribed in the first and second flow charts (100, 200) as describedabove. A program storage device (not shown) can perform the samefunctions as the program storage device 180 in FIGS. 5 and 6, and canprovide additional program instructions to other steps as needed.

Referring to steps 111 and 102, an aligned query list and an alignedtarget list are generated as described in the first flow chart 100.

Referring to steps 190, 303, 304, 305, 306, 301, 302, and 310collectively, a mass-to-charge ratio (m/z) tolerance parameter and aretention time (Rt) tolerance parameter that satisfy a specificationinput criterion for a false matching rate between the aligned query listand the target peak list. The generation of the m/z tolerance parameterand the Rt tolerance parameter that satisfy the specification inputcriterion can be generated by a single pass through steps 190 and 305,respectively, or can be generated by multiple passes through aniterative process.

Specifically, the generation of the m/z tolerance parameter and the Rttolerance parameter can be effected as follows. Referring to step 190, afalse matching rate is determined by matching the aligned query list andthe target peak list as described in the first flow chart 190. As aparameter for calculation of the false matching rate, an initialmass-to-charge ratio (m/z) tolerance is provided as an initial value fora mass-to-charge ratio tolerance parameter at step 303, and an initialretention time (Rt) tolerance is provided as an initial value for aretention time tolerance parameter at step 304.

Referring to step 305, the false matching rate that is calculated atstep 190 is compared with the specification input criterion for thefalse matching rate. The specification input criterion for the falsematching rate can be provided by a manual input step 301 for providing atarget false matching rate TFMR as an input and another manual inputstep 302 for providing a false matching rate tolerance FMRT. In thiscase, the specification range for the false matching rate is betweenTFMR−FMTR and TFMR+FMTR. The specification range for the false matchingrate is bound by 0 and 1. A specification range for the false matchingrate can be from 0.01 to 0.2, and typically from 0.03 to 0.1, althoughlesser and greater values for the specification range can be employed.The specification input criterion can be provided as a single range foracceptable values for the false matching rate, or can be a plurality ofspecification input criteria for the value of the false matching rate.At this point, an initial false matching rate between the aligned querylist and the target peak list is determined at step 305 employing theinitial m/z tolerance parameter and the initial Rt tolerance parameter.

At step 305, the algorithm of the third flow chart 300 causesdetermination of whether the initial false matching rate satisfies thespecification input criterion.

Referring to step 306, if the initial false matching rate does notsatisfy the specification input criterion, the initial m/z toleranceparameter and the initial Rt tolerance parameter are adjusted togenerate a revised m/z tolerance parameter and a revised Rt toleranceparameter, respectively. The revised m/z tolerance parameter is a newlyassigned value for the /z tolerance parameter, and the revised Rttolerance parameter is a newly assigned value for the Rt toleranceparameter. A systematic method for determining the values for therevised m/z tolerance parameter and the revised Rt tolerance parametercan be provided by program instructions from a program storage device(not shown). Any method for determining the values for the revised m/ztolerance parameter and the revised Rt tolerance parameter can beemployed, including, for example, comparison of the specification inputcriterion for the false matching rate and the value of the initial falsematching rate.

The process flow then proceeds to step 190. The algorithm of step 190performs a calculation to determine a revised false matching ratebetween the aligned query list and the target peak list employing therevised m/z tolerance parameter and the revised Rt tolerance parameter.The same algorithm is employed as at the first pass through step 190with different values for the m/z tolerance parameter and the Rttolerance parameter.

The steps 190, 305, and 306 are repeated until a revised false matchingrate, which is calculated at each additional pass through step 190,satisfies the specification input criterion for the false matching rate.The revised m/z tolerance parameter and the revised Rt toleranceparameter are cumulatively adjusted revised at each pass through step306. A revised false matching rate is determined employing a most recentrevised m/z tolerance parameter and a most recent revised Rt toleranceparameter at each pass through step 190. The step of adjusting therevised m/z tolerance parameter and the revised Rt tolerance parameter,i.e., step 306, and the step of determining the a revised false matchingrate, i.e., step 190, are repeated iteratively until the revised falsematching rate satisfies the specification input criterion as determinedat step 305.

If the false matching rate, whether it is an initial false matching rateor a revised false matching rate, satisfies the specification inputcriterion at step 305, the process flow proceeds to step 310. At step310, the most recent value of the m/z tolerance parameter and the mostrecent value of the Rt tolerance parameter are set as a new m/ztolerance parameter and a new Rt tolerance parameter, respectively. Them/z tolerance parameter and the new Rt tolerance parameter are initialvalues for an m/z tolerance parameter and an Rt tolerance parameter tobe employed at step 290.

Referring to step 290, a true matching rate between the aligned querylist and the target peak list is determined employing the m/z toleranceparameter and the Rt tolerance parameter, of which the values are set atstep 290. Steps 111 and 102 provide the aligned query list and thetarget peak list to step 290. The step 290 performs the same operationas in the second flow chart 200. A value for the true matching rate iscalculated at step 290 as described above.

Referring to step 320, the value for the true matching rate from step290 is compared with a stored value for the true matching rate from apreviously determination of the true matching rate. If the value for thetrue matching rate is greater than or equal to the stored value, theprocess flow proceeds to step 325. At step 325, the stored value for thetrue matching rate is updated with the value for the true matching rateas provided at step 290 in the most recent calculation, i.e., with thecurrent value for the true matching rate at step 320. At the same time,a value for the m/z tolerance parameter is stored as a “stored m/ztolerance parameter,” and a value for the Rt tolerance parameter isstored as a “stored Rt tolerance parameter.”

To insure that the value for the true matching rate at the first passthrough step 320 is stored as the stored value for the true matchingrate at step 325, the initial value for the true matching rate can beset to 0 for the purpose of comparison at step 320. Alternately, acounter for tracking the number of passes through step 320 can beemployed to trigger storage of the value for the true pass rate at step325 if the pass count is 1, i.e., for the first pass through step 320.

If the value for the true matching rate is lesser than the stored valuefor the true matching rate, which can be 0 for the first pass throughstep 320 or a value stored at a previous pass through step 325, theprocess flow proceeds to step 330. In this case, the stored value forthe true matching rate is not updated with the value. Correspondingly,the stored m/z tolerance parameter is not updated and the stored Rttolerance parameter is not updated. If the most recent value for thetrue matching rate is less than the stored value for the true matchingrate, the most recent values for the m/z tolerance parameter and the Rttolerance parameter are “inferior” to the stored values for the m/ztolerance parameter and the Rt tolerance parameter, i.e., the stored m/ztolerance parameter and the stored Rt tolerance parameter. In this case,the stored results from one of the previous passes through step 320provide a higher value for the true matching rate, i.e., provides a“superior” matching of peaks. Thus, the stored m/z tolerance parameterand the stored Rt tolerance parameter are preserved without a change.

Referring to step 330, the algorithm of an embodiment of the presentinvention determines whether an optimization search is complete. Thestep 330 can be performed immediately after step 320 if the stored m/ztolerance parameter and the stored Rt tolerance parameter are notupdated, or can be performed after step 325 if the stored m/z toleranceparameter and the stored Rt tolerance parameter are updated. Thedetermination on whether the optimization search is complete can bebased on a predefined criterion including at least one of a number ofiterations at the step of comparing the value with the stored value, ahistory of the stored value for the true matching rate, a history of thestored m/z tolerance parameter, and a history of the stored Rt toleranceparameter.

If the optimization search is determined to be complete at step 330, theprocess flow proceeds to steps 398 and 399 in parallel. At step 398, anoptimized m/z tolerance value is generated, which is the same as thecurrent value for the stored m/z tolerance parameter. At step 399, anoptimized Rt tolerance value is generated, which is the same as thecurrent value for the stored Rt tolerance parameter.

If the optimization search is determined to be incomplete at step 330,the process flow proceeds to step 310. At step 310, another value isassigned for each of the m/z tolerance parameter and the Rt toleranceparameter. The newly assigned values for the m/z tolerance parameter andthe Rt tolerance parameter can be generated by the program storagedevice (not shown) based on the stored values for the true matchingrate, the m/z tolerance parameter stored at step 325, and/or the Rttolerance parameter stored at step 325. Alternately or additionally, thenewly assigned values for the m/z tolerance parameter and the Rttolerance parameter can be provided as a manual input.

The process flow then proceeds to step 190 to determine a false matchingrate employing the m/z tolerance parameter and the Rt toleranceparameter as provided at step 310. The process flow then proceeds tostep 305 to determine whether the false matching rate as determined atstep 190 in the most recent pass satisfies the specification inputcriterion.

Depending on the outcome at step 305, the process flow can proceed tostep 310 or to step 306 at least once more. At step 306, at which them/z tolerance parameter and the revised Rt tolerance parameter areadjusted until the false matching rate satisfies the specification inputcriterion. The step of determining the true matching rate between thealigned query list and the target peak list employing the m/z toleranceparameter as adjusted and the Rt tolerance parameter as adjusted, i.e.,step 190, can also be repeated until the false matching rate satisfiesthe specification input criterion.

Referring to steps 320, 325, 330, 398, 399, 310, 190, 305, 306, 310, and290 collectively, an optimized m/z tolerance value and an optimized Rttolerance value are selected by repeating the step of selecting the m/ztolerance parameter and Rt tolerance parameter as performed at steps190, 305, and 310, and the step of determining the true matching rate asperformed at step 290. Further, the step of adjusting the revised m/ztolerance parameter and the revised Rt tolerance parameter, i.e., step306, and the step of determining a revised false matching rate until therevised false matching rate satisfies the specification input criterion,i.e., steps 190 and 205 collectively, can be repeated to select theoptimized m/z tolerance value and the optimized Rt tolerance value.

Referring to FIG. 8, a fourth flow chart 400 illustrates a method forgenerating an optimal list of matches once the optimal m/z tolerancevalue and the optimal Rt tolerance value are determined. Referring tostep 460, peaks are matched between the aligned query list from step 111and the target peak list from step 102 employing the method of PPMdescribed above. The optimal m/z tolerance value from step 398 and theoptimal Rt tolerance value from step 399 are employed as a matchingparameter for PPM.

Referring to step 490, an optimal list of matches among peaks across thealigned query list and the target peak list is generated employing theoptimized m/z tolerance value and the optimized Rt tolerance value asmatching parameters. The optimal list of matches is the output of thealgorithm according to an embodiment of the present invention. Theoptimal list of matches includes a dataset on peaks. The dataset onpeaks includes at least a calculated m/z and Rt for each peak.

Referring to FIG. 9, an exemplary system 900 according to the presentinvention is shown. The exemplary system 900 can be employed formatching peaks in liquid chromatography-mass spectroscopy (LC-MS)datasets from multiple runs. The exemplary system includes a computingdevice that is configured to perform program instructions. The computingdevice can include a memory and a processor device in communication withthe memory. The program instructions can configure the computing deviceto perform the steps of embodiments of the present invention describedabove. The exemplary system 900 can be a computer-based system in whichthe methods of the embodiments of the invention can be carried out by anautomated program of machine-executable instructions to match peaks inliquid chromatography-mass spectroscopy (LC-MS) datasets from multipleruns.

A data storage device that is programmable and readable by a machine andtangibly embodying or storing a program of instructions that areexecutable by the machine to perform the methods described herein arealso provided. For example, the automated program can be embodied, i.e.,stored, in a machine-readable data storage devices such as a hard disk,a CD ROM, a DVD ROM, a portable storage device having an interface suchas a USB interface, a magnetic disk, or any other storage mediumsuitable for storing digital data.

The computer-based system includes a processing unit 910, which is acomputing device and houses a processor device, a memory and othersystems components (not shown expressly in the drawing) that implement ageneral purpose or special purpose processing system, or computer thatcan execute a computer program product. The computer program product cancomprise media, for example a compact storage medium such as a compactdisc, which can be read by the processing unit 910 through a disc drive920, or by any means known to the skilled artisan for providing thecomputer program product to the general purpose processing system forexecution thereby. The exemplary system 900 can include an LC-MSapparatus 905 and/or an LC-MS database. Alternately, the LC-MS apparatus905 and/or an LC-MS database 121 can be external to the exemplary system900 and electronically connected through a wired network or a wirelessnetwork.

The computer program product can comprise all the respective featuresenabling the implementation of the inventive method described herein,and which is able to carry out the method when loaded in a computersystem. Computer program, software program, program, or software, in thepresent context means any expression, in any language, code or notation,of a set of instructions intended to cause a system having aninformation processing capability to perform a particular functioneither directly or after either or both of the following: (a) conversionto another language, code or notation; and/or (b) reproduction in adifferent material form.

The computer program product can be stored on hard disk drives withinthe processing unit 910, as mentioned, or can be located on a remotesystem such as a server 930, coupled to the processing unit 910, via anetwork interface such as an Ethernet interface. A monitor 940, a mouse950 and a keyboard 960 are coupled to the processing unit 910, toprovide user interaction. A scanner 980 and a printer 970 can beprovided for document input and output. The printer 970 is shown coupledto the processing unit 910 via a network connection, but can be coupleddirectly to the processing unit 910. The scanner 980 is shown coupled tothe processing unit 910 directly, but it should be understood thatperipherals might be network coupled, or direct coupled withoutaffecting the ability of the processing unit 910 to perform the methodof the invention.

While the present invention has been particularly shown and describedwith respect to preferred embodiments thereof, it will be understood bythose skilled in the art that the foregoing and other changes in formsand details can be made without departing from the spirit and scope ofthe present invention. For example, variations that combine varioussteps of the first, second, and third exemplary programs in a singleprogram can be employed. It is therefore intended that the presentinvention not be limited to the exact forms and details described andillustrated, but fall within the scope of the appended claims.

1. A non-transitory machine-readable data storage device embodying aprogram of machine-executable instructions to match peaks in liquidchromatography-mass spectroscopy (LC-MS) datasets from multiple runs,wherein said program comprises steps of: generating, by employing aprocessor and a memory in communication with said processor, an alignedquery list for peaks from a first dataset from a first LC-MS run;generating, by employing said processor and said memory, a target peaklist for peaks from a second dataset from a second LC-MS run;generating, by employing said processor and said memory, amass-to-charge ratio (m/z) tolerance parameter and a retention time (Rt)tolerance parameter that satisfy a specification input criterion for afalse matching rate between said aligned query list and said target peaklist; determining, by employing said processor and said memory, a truematching rate between said aligned query list and said target peak listemploying said m/z tolerance parameter and said Rt tolerance parameter;selecting, by employing said processor and said memory, an optimized m/ztolerance value and an optimized Rt tolerance value by repeating saidstep of selecting said m/z tolerance parameter and Rt toleranceparameter and said step of determining said true matching rate; andgenerating, by employing said processor and said memory, an optimal listof matches among peaks across said aligned query list and said targetpeak list employing said optimized m/z tolerance value and saidoptimized Rt tolerance value as matching parameters.
 2. Thenon-transitory machine-readable data storage device of claim 1, whereinsaid program further comprises steps of: fetching, by employing aprocessor and a memory in communication with said processor, said firstdataset from an LC-MS apparatus or an LC-MS database; and fetching, byemploying said processor and said memory, said second dataset from saidLC-MS apparatus, another LC-MS apparatus, said LC-MS database, oranother LC-MS database.
 3. The non-transitory machine-readable datastorage device of claim 1, wherein said step of generating said m/ztolerance parameter and said Rt tolerance parameter comprises steps of:determining an initial false matching rate between said aligned querylist and said target peak list employing an initial m/z toleranceparameter and an initial Rt tolerance parameter. determining whethersaid initial false matching rate satisfies said specification inputcriterion; and if said initial false matching rate does not satisfy saidspecification input criterion, adjusting said initial m/z toleranceparameter and said initial Rt tolerance parameter to generate a revisedm/z tolerance parameter and a revised Rt tolerance parameter,respectively, and determining a revised false matching rate between saidaligned query list and said target peak list employing said revised m/ztolerance parameter and said revised Rt tolerance parameter.
 4. Thenon-transitory machine-readable data storage device of claim 3, whereinsaid step of generating said m/z tolerance parameter and said Rttolerance parameter comprises steps of: adjusting said revised m/ztolerance parameter and said revised Rt tolerance parameter; determininganother revised false matching rate employing a most recent revised m/ztolerance parameter and a most recent revised Rt tolerance parameter;and repeating said step of adjusting said revised m/z toleranceparameter and said revised Rt tolerance parameter and said step ofdetermining said another revised false matching rate until said anotherrevised false matching rate satisfies said specification inputcriterion.
 5. The non-transitory machine-readable data storage device ofclaim 1, wherein said program further comprises a step of comparing avalue for said true matching rate from said step of determining saidtrue matching rate with a stored value for said true matching rate froma previously determination of said true matching rate, said stored valuebeing stored in a data storage medium.
 6. The non-transitorymachine-readable data storage device of claim 5, wherein said programfurther comprises, if said value for said true matching rate is greaterthan said stored value, steps of: updating said stored value with saidvalue for said true matching rate; storing in said data storage medium avalue for said m/z tolerance parameter as a stored m/z toleranceparameter; and storing in said data storage medium a value for said Rttolerance parameter as a stored Rt tolerance parameter.
 7. Thenon-transitory machine-readable data storage device of claim 6, whereinsaid program further comprises steps of: determining whether anoptimization search is complete after comparing said value with saidstored value based on a predefined criterion comprising at least one ofa number of iterations at said step of comparing said value with saidstored value, a history of said stored value for said true matchingrate, a history of said stored m/z tolerance parameter, and a history ofsaid stored Rt tolerance parameter; if said optimization search iscomplete, generating said optimized m/z tolerance value and saidoptimized Rt tolerance value, wherein said optimized m/z tolerance valueis said stored m/z tolerance parameter and said optimized Rt tolerancevalue is said stored Rt tolerance parameter.
 8. The non-transitorymachine-readable data storage device of claim 7, wherein said programfurther comprises conditional steps of: assigning another value for saidm/z tolerance parameter and another value for said Rt toleranceparameter; determining another false matching rate employing said m/ztolerance parameter and said Rt tolerance parameter; and determiningwhether said another false matching rate satisfies said specificationinput criterion.
 9. The non-transitory machine-readable data storagedevice of claim 8, wherein said conditional steps further comprise:adjusting said m/z tolerance parameter and said revised Rt toleranceparameter until said another false matching rate satisfies saidspecification input criterion; and repeating said step of determiningsaid true matching rate between said aligned query list and said targetpeak list employing said m/z tolerance parameter as adjusted and said Rttolerance parameter as adjusted.
 10. The non-transitory machine-readabledata storage device of claim 1, wherein said step of determining saidtrue matching rate comprises a step of splitting said aligned query listinto an annotated query list and a non-annotated query list.
 11. Thenon-transitory machine-readable data storage device of claim 10, whereinsaid step of determining said true matching rate comprises a step ofsplitting said target peak list into an annotated target list and anon-annotated target list.
 12. The non-transitory machine-readable datastorage device of claim 11, wherein said step of determining said truematching rate comprises a step of matching peaks between said annotatedquery list and said annotated target list.
 13. The non-transitorymachine-readable data storage device of claim 12, wherein said step ofdetermining said true matching rate further comprises a step ofgenerating a true match list based on said matching of peaks betweensaid annotated query list and said annotated target list, wherein saidtrue matching rate is determined from said true match list.
 14. Thenon-transitory machine-readable data storage device of claim 13, whereinsaid true matching rate is a ratio of a total number of matches thatdoes not require a mass shift to achieve a match to a total number ofmatches in peaks.
 15. The non-transitory machine-readable data storagedevice of claim 1, wherein said step of generating said m/z toleranceparameter and said Rt) tolerance parameter comprises a step ofgenerating a shifted target list by mass-shifting peaks in said targetpeak list.
 16. The non-transitory machine-readable data storage deviceof claim 15, wherein said step of generating said m/z toleranceparameter and said Rt) tolerance parameter further comprises a step ofmatching peaks between said aligned query list and said shifter targetlist.
 17. The non-transitory machine-readable data storage device ofclaim 16, wherein said step of generating said m/z tolerance parameterand said Rt) tolerance parameter further comprises a step of generatinga false match list based on said matching of peaks between said alignedquery list and said shifter target list, wherein said false match rateis calculated based on said false match list.
 18. The non-transitorymachine-readable data storage device of claim 17, wherein said falsematching rate is a ratio of a total number of matches that requires amass shift to achieve a match to a total number of matches in peaks. 19.The non-transitory machine-readable data storage device of claim 18,wherein said mass-shifting of said peaks in said target peak list shiftssaid peak by a mass between 3 Da and 200 Da.
 20. The non-transitorymachine-readable data storage device of claim 1, wherein said optimallist of matches comprises data on peaks, said data on peaks comprisingat least a calculated m/z and Rt for each peak.